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EDITORIAL FOCUS
Department of Cell Biology, University of Virginia Health System, Charlottesville, Virginia
THE MEMBRANES OF EUKARYOTIC cells contain small amounts of phosphoinositides, which are phosphorylated forms of the membrane lipid phosphatidylinositol (PtdIns). Together, PtdIns and its phosphoinositide derivatives constitute 
10% of the total cellular lipid in most cells. Although phosphoinositides were first recognized as second messengers in a variety of signal transduction cascades, it has become increasingly apparent that they also serve as important regulators of vesicular transport.
PtdIns can be phosphorylated at the D-3, D-4, or D-5 positions of the inositol ring, generating seven distinct phosphoinositide species: PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. Each of these phosphorylated derivatives has a distinct set of biological activities that are typically mediated by proteins that recognize and selectively bind to the phosphorylated head group. Several different phospholipid-binding domains have been characterized, including PH domains, which bind a variety of phosphoinositide species, PX domains, which have been shown to bind PtdIns(3)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, ANTH/ENTH domains, which bind PtdIns(4)P, PtdIns(5)P, PtdIns(4,5)P2, or PtdIns(3,5)P2, and FYVE domains, which appear to selectively bind PtdIns(3)P (15, 20).
Phosphorylation at the D-3, D-4, and D-5 positions is carried out by distinct classes of PtdIns kinases that are differentially localized within cells; therefore, different phosphoinositide species are concentrated in different membrane compartments. For example, PtdIns(4)P is relatively abundant in the Golgi apparatus, PtdIns(3)P is concentrated on endosomal membranes and PtdIns(4,5)P2 is most abundant at the plasma membrane. Although inferred from the localization of the enzymes that catalyze their synthesis, the distribution of these different phosphoinositides has been confirmed by imaging of green fluorescent protein fusions containing phosphoinositide-specific binding domains (for review, see Ref. 11).
Phosphoinositides in endocytic transport. Degradation of membrane proteins and lipids is an important aspect of membrane homeostasis in eukaryotic cells. The steady-state distribution of plasma membrane proteins is achieved by balancing the delivery of newly synthesized components from the Golgi with the recycling of some internalized components back to the plasma membrane and the degradation of others in lysosomes. In addition to the constitutive turnover of membrane constituents, the ligand-triggered degradation of signaling receptors, such as receptor tyrosine kinases and G protein-coupled receptors, is an important mechanism for signal downregulation. However, the business end of such receptors (e.g., kinase domains and/or binding sites for associated signal transducers) is invariably located in the cytoplasm, posing a topological problem: the signaling domain must be translocated to the endosomal lumen so that it is both sequestered from the cytoplasm and is exposed to lysosomal hydrolases along with the rest of the molecule.
Early experiments following the transport of EGF receptors indicated that both EGF (10) and the receptor itself (7) are sorted into the lumen of multivesicular bodies (MVBs), a subset of late endosomes whose lumen is filled with small vesicles. Fusion of MVBs with lysosomes (or their maturation into lysosomes) thus exposes both the vesicles and their associated proteins to the lysosomal enzymes that mediate their degradation (7). Studies in both yeast and mammalian systems have identified a complex machinery for the sorting of membrane proteins into the interior of multivesicular bodies. Three Endosomal Complex Required for Transport (ESCRT) complexes (ESCRT I, II, and III) mediate the sequential recognition, sorting, and import of ubiquitylated cargo proteins into the internal vesicles of the MVB (reviewed in Ref. 1). In addition to these protein components, phosphoinositides have also been implicated in this process. PtdIns3-P is enriched in the membranes of both early endosomes and MVBs (9), and appears to be necessary for both the sorting of cargo into the MVB lumen and the formation of internal vesicles, but not for the biogenesis of late endosomes per se (19). If PtdIns-kinase activity is blocked pharmacologically, or if PtdIns(3)P is sequestered by overexpression of recombinant FYVE domains, ubiquitylated cargo (e.g., EGFR) is retained in the limiting membranes of endosomes (19).
A second phosphoinositide implicated in this process is PtdIns(3,5)P2. In yeast, this species is generated by a single enzyme, Fab1p, which converts PtdIns(3)P to PtdIns(3,5)P2 (8). Fab1p contains an NH2-terminal FYVE domain through which it binds endosomal PtdIns(3)P and a COOH-terminal lipid kinase domain. Related proteins have been identified in Drosophila, Caenorhabditis elegans, Arabidopsis, and all vertebrates indicating that it is highly conserved (21). The mouse and human orthologs of Fab1p have been called PIKfyve (26, 3) or PIPKIII (17). In yeast, Fab1p activity is necessary for the sorting of MVB cargo and the formation of MVB-like structures, and deletion of the FAB1 gene results in a swollen and poorly acidified vacuole (18). Importantly, mouse PIKfyve can restore normal vacuolar morphology to 
fab1 yeast strains, confirming that it is indeed a functional ortholog of Fab1p (16). In addition, expression of a catalytically inactive PIKfyve mutant induces similar phenotypic effects in mammalian cells, causing swelling of late endosomes and a reduction in the number of luminal vesicles in MVBs (12). An excellent review on the subject of PtdIns(3,5)P2 can be found in Ref. 17.
Unlike other phosphoinositides, whose localization can be determined by either specific antibodies or reporter constructs (11), no reagents of sufficient specificity currently exist for PtdIns(3,5)P2. Therefore, the distribution of this species in cells has been inferred from the localization of either endogenous or exogenously expressed PIKfyve (reviewed in Refs. 25 and 20). Because PIKfyve localizes primarily to late endosomes/MVBs at steady state, and PIKfyve mutants cause morphological alterations in this compartment, it has been widely assumed that PIKfyve functions primarily in late endosomes and that PtdIns(3,5)P2 would be concentrated there as a result.
In the present issue of AJP–Cell Physiology, Ikonomov et al. (Ref. 14; see p. C393 of this issue) report that PIKfyve may actually function earlier in the endocytic pathway than has been previously thought. As noted above, previous work has shown that expression of a catalytically inactive murine PIKfyve mutant (K1831E) leads to the formation of enlarged vacuolar structures, many of which contain late endosomal markers (17, 15). However, the number of vacuoles and their size apparently increases with the duration of expression to the point where cells fill with swollen endosomes that eventually fuse, thus occupying a large fraction of the cytoplasmic volume. In the present study, the authors carefully examined cells at short times after transfection with expression plasmids encoding either wild-type PIKfyve or the catalytically inactive K1831E mutant. As previously reported (13), the wild-type protein localized primarily to perinuclear (presumably late) endosomes and did not cause any obvious changes in endosomal morphology. In contrast, the catalytically inactive mutant localized to more peripheral structures that also labeled for the early endosomal marker proteins rab5 and EEA1 (14). This localization was dependent on the presence of active rab5 and PtdIns(3)P on the endosomal membranes, consistent with the notion that rab5 promotes the recruitment of the PtdIns 3-kinases responsible for synthesis of PtdIns3-P on endosomal membranes (3).
The presence of the inactive PPIKfyve mutant induced the swelling of early endosomes, which could in principle be due to either enhanced endosome/endosome fusion or to decreased budding of carrier vesicles from endosomal membranes. Using in vitro fusion assays, the authors demonstrated that cytosol containing catalytically inactive PIKfyve promoted endosome/endosome fusion, while cytosol containing wild-type PIKfyve actually inhibited fusion (14). Because endosomal PtdIns(3)P is necessary for the recruitment of EEA1, an essential component of the fusion reaction (22), these observations suggest that PtdIns(3)P is depleted by wild-type PIKfyve through conversion to PtdIns(3,5)P2. In contrast, PIKfyveK1831E impairs this conversion, resulting in the accumulation of PtdIns(3)P on endosomal membranes and enhanced membrane fusion. It should be noted that Stenmark and colleagues recently reported that wild-type human PIKfyve localizes at least partially to early endosomes at steady state (2). The basis for the differential localization of mouse and human forms of PIKfyve is unknown, but could be due to differences in cell type or the level of ectopic expression.
How can we incorporate these observations into current models of postendocytic membrane transport? First, they suggest that PIKfyve activity is not only necessary for the maintenance and function of late endosomal compartments, but for early endosomes as well. In retrospect, it is surprising that so little PIKfyve is associated with early endosomes at steady state because its FYVE domain is functionally similar to FYVE domains on EEA1 and Hrs, two proteins that do concentrate on early endosomal membranes. One reasonable hypothesis supported by the data is that PIKfyve is transiently recruited to early endosomes via interaction of its FYVE domain with PtdIns(3)P, but that its rapid local conversion of PtdIns(3)P to PtdIns(3,4)P2 creates membrane microdomains that can no longer support PIKfyve binding. Such microdomains may have an important function in the subsequent biogenesis of MVBs through the recruitment of downstream effector proteins. One protein that has been reported to bind PtdIns(3,5)P2 is the epsin-like protein Ent3p. Ent3p binds PtdIns(3,5)P2 via its ENTH domain, and a point mutation that disrupts this interaction interferes with MVB formation in yeast (6). Intriguingly, Ent3p (and the related mammalian protein epsinR) binds clathrin, AP1, and the monomeric GGA adaptor proteins (5), suggesting that it promotes vesicle formation on endosomal membranes. Such carrier vesicles may be important in the removal of recycling membrane components during the maturation process from early endosome to MVB.
Second, it is likely that invagination of vesicles into the endosomal lumen begins as early endosomes mature, but before they are morphologically recognizable as MVBs. It is doubtful that Ent3p or epsinR participate in this process, because clathrin-coated vesicles evaginate toward the cytoplasm. However, interfering with PIKfyve activity reduces the number of intralumenal vesicles in the resulting aberrantly formed MVBs (12), suggesting that PtdIns(3,5)P2 may have an additional, as yet undefined role in vesicle invagination.
Compared with early endosomes, late endosome/MVBs are relatively poor in PtdIns(3)P. Why then is PIKfyve concentrated on these compartments at steady state? One possible explanation is that additional protein-protein interactions stabilize the association of PIKfyve with these compartments. One potential binding partner is Vac14p (and its human ortholog hVac14), which allosterically activate PIKfyve, and localize to similar compartments (2, 5, 23). A second potential partner is Svp1p, which facilitates the recycling of marker proteins from the yeast vacuole to the Golgi (4). Several related proteins have been identified in both yeast and mammals, which may have redundant or overlapping functions.
Clearly PIKfyve is an important regulator of postendocytic transport in eukaryotic cells. Challenges for the future will include: 1) the generation of reporter molecules that can be used to localize PtdIns(3,5)P2 in intact cells, 2) defining the role of PtdIns(3,5)P2 in intralumenal vesicle formation, 3) defining the function of Ent3p and related molecules in endosome maturation, and 4) identifying and characterizing additional PtdIns(3,5)P2 effector proteins, such as Svp1p.
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FOOTNOTES |
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